The migratory locust is an important agricultural test. This method provides an efficient and easy to yield protocol for the gene pressure methods of the It is a systematic and a detailed method of a CRISPR/CAS9 construction of homozygous locust mutants, including egg collection, mutant screening and the homozygous maintaining. To begin, prepare the injection needles by pulling the glass capillaries with a micro pipette puller.
Set the parameters as described in the text manuscript, then grind the tip of the injection needle with a micro grinder. Prepare ribonucleo protein for the injection by mixing one microliter of Cas9 protein with one microliter of the verified single guide RNA. Then add eight microliters of RNA's free sterile water to make the final volume of the mixture, 10 microliters.
Mix the solution thoroughly and place it on ice. Next, collect the freshly laid egg pods from the ova position pod, and wait for egg tanning. After 30 minutes, use a fine brush to isolate the eggs from the egg pods in water, and wash the eggs three times with sterile water.
Then place the eggs in a Petri dish and add sterile water to keep them moist. Fill the injection needle with the prepared ribonucleo protein solution, and load the injection to the micro manipulator. Set the microinjection parameters as 300 Hectopascals of injection pressure, 0.5 seconds of injection time and 25 Hectopascals of compensation pressure.
When the parameters are set, press the pedal to evaluate the volume of the injected solution. Adjust the injection pressure and injection time to ensure that the injection volume is about 50 to 100 nanoliters. After arranging the sterile eggs regularly on the injection pad, place the pad on the object table of the microscope and adjust the magnification of the microscope until the eggs are observed.
Then adjust the microinjection needle under the microscope until the injection tip can be seen and position the tip near the egg to be injected. Start the injection at a suitable height and an angle of 30 to 45 degrees. Insert the tip into the egg near the micro piles of the egg and press the pedal to accomplish the injection.
Next, retract the needle quickly and move the injection pad for injection of the next egg. Transfer the injected eggs into a culture dish with a piece of moist filter paper. Place them in an incubator at 30 degrees Celsius.
After injection, check the development of the injected eggs every day. Transfer the injected eggs to a new culture dish every 24 hours in the first five days. On the sixth day after the injection, randomly pick and transfer 10 eggs into a tube and adequately grind the eggs with two steel balls at 60 hertz for six minutes using a grinder.
After grinding, resuspend the debris in one milliliter PBS. Then transfer five microliters of the resuspended mixture into 45 microliters of 50 millimolar sodium hydroxide to lyse at 95 degrees Celsius for five minutes. To terminate the alkaline lysis reaction, add five microliters of one molar Tris hydrochloride solution into the lysis system.
Take one microliter of the lysis product as the PCR template to amplify the target gene fragment. After the PCR and sequencing, compare the sequencing results with the wild type sequence to evaluate whether the CRISPR/Cas9 system cleaved the target gene in vivo. Allow the rest of the eggs for subsequent development as indels are detected at the embryonic stage.
After development, transfer and culture the hatched nymphs into a rearing cage as described in the manuscript. When the nymphs develop to the fifth instar stage, separate them with plastic culture cups. Perform cross-breeding using the G0 mutants and wild type locusts.
After collecting oassists, use three to six developed eggs in each oassist to detect mutations as described earlier. Keep mutation positive oassists for the subsequent development and abandon the mutation negative oassists. For cryo-preservation, wash the eggs with sterile water and incubate them in a culture dish as explained before.
After five to six days, gather the eggs together in the culture dish, and cover them with filter paper fragments. Then wrap the entire culture dish with a paraffin film. Place the dish at 25 degrees Celsius for two days, followed by another two days at a lower temperature of 13 to 16 degrees Celsius.
Then transfer the dish to a six degree Celsius refrigerator. Add water to the dish every two weeks to provide a moist environment for the eggs. Finally, for resuscitating the cryopreserved eggs, place the Petri dish at 25 degrees Celsius for two days.
Later, place the eggs in a 30 degrees Celsius incubator until the nymphs hatch. In this study, the target site for the CRISPR/Cas9 system was located in the first exon. In the Cas9 cleavage assay, the CRISPR/Cas9 ribonucleo protein could digest the PCR fragment containing the target site with a cleavage rate of about 55%The sequencing results for embryonic stage mutation rate estimation suggested efficient genome editing at the target site.
The editing efficiency resulted in a 52.73%nymph hatching rate and 66.67%of the G0 adults were mosaic mutants. The passaging strategy of the mutant lines is shown here. The excess homozygous eggs were cryopreserved to improve the utilization rate of the homozygotes.
Although the hatching rate of the cryopreserved eggs was reduced with the extension of cryopreservation time, the remedial actions such as applying filter paper fragments and recovering these preserved eggs with a gradient of rising temperature were helpful for the resuscitation. The most important thing is to try to limit the mechanical damage caused by microinjection. This procedure can be used as a reference for the attaining other insects.
Part of some details such as methods of egg collection, injection and the crop should be modified. This technique can also be helpful to study metamorphosis and the environmental adaptation of the insects and the construction of homozygous locust mutants accelerated the research in this field.
